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dear all,
I recently developed a method & preformed haemolysis effect as per guideline. i observed 50% Haemolyis effect on highly haemolysed plasma lots having K2EDTA. although performed matrix effect with same haemolysed plasma lots, no ME observed. even in human plasma samples (haemolysed partially), no haemolysis effect found. As deutoriated IS is used haemolysis QC's are passing against freshly prepared CC std.
how can we remove haemolysis effect?
In method OPA was used to break binding.
Ashish saxena
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what is the 50% hemolysis effect? Where does the hemolysis occur, on draw, on transfer, on standing? Like almost everything else it is probably easier to avoid hemolysis than to compensate for hemoslysis later. Iron and copper and O2 are good oxidants.
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Dear efoconnor,
thanks for reply sir,
Human plasma samples that we receive for analysis from clinical deptt., about 2% get haemolyed. we have to analysis all the sample proving that there is no haemolyisis effect. During ananlysis it is observed response of drug is less that that of compared with non-haemolysed samples. May be due to the protein binding is more in haemolysed samples. So in bioananlyis how can we break this type of binding to recover more drug.
ashish saxena
associate scientist
lupin bioresearch centre
india
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Dear Ashish,
You have opened an interesting chapter of bioanalysis. The latest trends and research is now going on to avoid hemolysis effects and matrix effects especially when bioanalysis is done by LCMSMS.
I would recommend you to first find out whether it is due to hemolysis or due to ion suppression.
After LCMSMS came to the picture, people are giving less concentration in chromatography part and that is where we are lagging.
The only way to ovoid such problems is to develop good chromatography. You have to develop your method so that you can avoid matrix effect and hemolysis effect. After method development, please confirm your method by performing one or two P& A batches (CC should be in normal plasma and QCs should be in hemolysed plasma).
You can try gradient method, which works fine in such cases but you have to compromise your total run time in such cases. You can try SPE extraction procedure especially with phospholipids removal cartridges, available with Orochem. This cartridge (Column) really works. You can try- definitely we will be surprise. But don't forget to optimize your chromatography.
Regards,
S.Basu
Senior Research Scientist (DMPK)
Sai Advantium Pharma Ltd.,Pune
E-Mail : sudipta.b.at.saiadvantium.com
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You could also try to limit or prevent hemolysis pre-analytically.
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Dear Sudipta,
Thanks for reply
I have checked the matrix effect using Haemolysed plasma lots in method development. Even it is not recommened in guidelines to check ME in haemolysed lots. NO matrix effect observed in Normal as well as in haemolysed plasma samples having same anticoagulent (K2EDTA). So it is confirmed that there is no matrix effect in haemolysed plasma samples.
Haemolysis effect: As i prepared cc with normal plasma Lot and QC's in different Lots of haemolysed plasma. All QC's are passing because i m using deutoriated IS for analyte, even the response is less than 50% for both analyte and IS.
This is first time i observed haemolysis effect having no ME/ion suppression in human plasma samples.
I optimized a good chromatography and proved simulated run of a batch size if 250 samples, run time 3.5 min i.e. 150*3.5=15 hrs batch. Not a single QC is failing.
During extraction drug is not degrading by extraction solvents as normal plasma samples are passing.
SO WHAT IS HAPPENING WITH HAEMOLYSED SAMPLES?
Ashish Saxena
Associate scientist
Lupin Bioresearch Centre
ashishsaxena.-a-.lupinpharma.com
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Dear Ashish,
I believe, you had a tough time. But relax it's a part of bioanalyst. I'm sure you will definitely succeed.
What I have understand, it is a clear cut matrix effect problem. May be with few matrices (what you have checked) you didn't observed the effect.
Can you share me how you did your matrix effect experiment?
You can also try the Orochem's phospholipids removal cartridges during extraction.
Regards,
Sudipta Basu
Senior Research Scientist (DMPK)
Sai Advantium Pharma, Pune
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